Structural And Functional Studies Of Glycolate Oxidase(GOX) From Nicotiana Benthamiana

It is well known that photosynthesis is closely related to agricultural production.The net photosynthetic rate is reduced by 25%-50%for carbon metabolism because of simultaneous photorespiration in plant cells.Glycolate oxidase(GOX)is a flavin mononucleotide(FMN)-dependent a-hydroxy acid-oxidizing protein that oxidizes glycolate to glyoxylate at the expense of O2 with the production of H2O2,which is one key step in photorespiration.In human,GOX is located in liver peroxisomes,catalyzing the FMN-dependent oxidation of glycolate to glyoxylate and oxalate.The ability of GOX to produce oxalate,a key metabolite in the formation of kidney stone,leads to the liver oxalate overproduction in patients with primary hyperoxalaria type I.In recent years,GOX has been selected as a safe and efficient target in treating primary hyperoxaluria and kidney stones.However,there is no structure information of GOX protein without its cofactor FMN in the Protein Data Bank database up to date,and the molecular mechanism of how GOX carries out its catalytic function and the conformational changes in the catalytic process are unclear.To understand the details of the catalytic mechanism,we have focused on the GOX from Nicotiana benthamiana(NbGOX)and determined two structures of NbGOX with or without cofactor FMN(named Holo-GOX and Apo-GOX,respectively).The results show that GOX purified by neutral buffer(Holo-GOX)contains FMN and has strong enzyme activity,while GOX purified by partially alkaline(Apo-GOX)buffer contains no FMN and basically loses enzyme activity.The two protein structures both appear a(?/?)8-barrel type consisting of eight ?-strands inside and eight a-helices outside.In Holo-GOX,FMN is located at the C-terminal of ?-strands of(?/?)8-barrel,combining with GOX by hydrogen bonds.Loop 6,loop 4 and the loop between aB-aC form a "lid" to isolate FMN from the solution and wrap it in the(?/?)s barrel structure to prevent the dissociation of FMN.In Apo-GOX,both loop 6 and the loop between aB-aC swing toward the solution.Loop 4 becomes more flexible and the aD disappears.These changes lead to the opening of the "lid" and the dissociation of FMN from GOX.The charge in the pocket of the active center also changes with the movement of structure.Although Holo-GOX and Apo-GOX both exist as tetramers in the solutions and crystals,the structure of Apo-GOX tetramer is looser.Structural information indicates that each lid of holo-GOX protomer has strong interactions like hydrogen bonds and salt bridges with the neighboring GOX.These interactions make up an inter-molecular pH sensor.When purified buffer changes from neutral to partially alkaline,we infer that the inter-molecular interactions in pH sensor,such as hydrogen bonds and salt bridges,are reduced or even disrupted.Then the lid formed by loop6,loop4 and loop between aB-aC transforms from closed conformation to open conformation.The GOX protomers rotate slightly,decreasing the area of interface and creating a novel binding surface formed by their C-terminus during the process.After comparing w:ith the structures of the weak homologous human GOX,we find that the inter-molecular pH sensor also exists.By analyzing the crystal structures of Holo-GOX and Apo-GOX,we provide a new insight in the molecular mechanism of how pH affects the GOX biological activity through binding FMN.The research on GOX has important theoretical significance and practical application value in the current situation of increasing population and decreasing cultivated land in the world.And it also provides a new direction in the treatment of primary hyperoxaluria and kidney stones.